Method for detecting acetylated PD-L1 in cell lysates

Place supplemented Bead Wash Buffer on ice.

LYSATE PREPARATION: BUFFERS AND EQUIPMENT

Determine the volume of lysis and dilution buffer needed to process your cells.

For each 1 mg of lysate protein you will need:

                                                                                300 μl    supplemented Lysis Buffer

                                                                                1.2 ml    supplemented Dilution Buffer

Make buffers as detailed below just prior to processing cell lysates. Buffer volumes should be scaled according to needs, the recipes are for 1 ml of each buffer. Each 1ml of Lysis buffer requires 4 ml of Dilution buffer.

Supplemented Lysis Buffer (1 ml): Store at room temperature, use within 3h

A	B	C	D
BlastR Lysis Buffer	1X	1 ml	1X
Halt Protease Inhibitor Cocktail*	100X	10 μl	1X
Trichostatin A	5 mM	0.4 μl	2 μM
Nicotinamide	2 M	20 μl	40 μM
N-ethylmaleimide**	1 M	10 μl	10 μM

*This inhibitor cocktail can be replaced with the following mix of protease inhibitors; pepstatin A (10 μM final), leupeptin (50 μM final), aprotinin (2 μg/ml final), bestatin (40 μM final)**N-ethylmaleimide is included as a deubiquitinase (DUB) inhibitor as we are studying acetylation/ubiquitination of PD-L1, we have not tested this assay in the absence of N-ethymaleimide.

Supplemented Dilution Buffer (1 ml): Store on ice, use within 3h

A	B	C	D
BlastR Lysis Buffer	1X	1 ml	1X
Halt Protease Inhibitor Cocktail*	100X	10 μl	1X
Trichostatin A	5 mM	0.4 μl	2 μM
Nicotinamide	2 M	20 μl	40 μM
N-ethylmaleimide	1 M	10 μl	10 μM

 *This inhibitor cocktail can be replaced with the following mix of protease inhibitors; pepstatin A (10 μM final), leupeptin (50 μM final), aprotinin (2 μg/ml final), bestatin (40 μM final)

1:4 Dilution Buffer (1 ml): Store on ice, use within 3h

A	B
200 μl	800 μl

Reagents and Equipment

Phosphate Buffered Saline (PBS) pH 7.0

Microfuge tubes

Cell lifter

BlastR filters (each filter can process up to 1 ml of lysed cells)

Liquid nitrogen (to snap freeze lysates)

CELL LYSATE PREPARATION: PROCESSING CELL LYSATES

The required number of cells should be grown and treated as required (see Before Starting section above).

Remove culture media and wash cells twice with 10 ml each of room temperature PBS buffer.

Add the appropriate volume of Supplemented Lysis Buffer determined in STEP 1 and harvest cells using a cell lifter. The lysate will become highly viscous, due to nuclear lysis. If processing multiple plates for a given condition, the lysates can be pooled at this point (use a snipped 1 ml pipette tip).

Use a snipped 1 ml pipette to transfer up to 1 ml of crude lysate per BlastR filter and harvest the flow through into a 15 ml tube on ice. The filter step is necessary to reduce sample viscosity (by removing DNA from the lysate).

Record the volume of the lysate and add 4 volumes of Supplemented Dilution Buffer. Mix gently.

Immediately take the protein concentration.

Dilute the lysate to 1 mg/ml using 1:4 Dilution Buffer and immediately dispense the lysate into 1 mg aliquots.

Snap freeze the lysate aliquots in liquid nitrogen and store at -80°C.

ACETYL-LYSINE IP: BUFFERS AND EQUIPMENT

Bead Wash Buffer: 100 ml, store at room temp.

A	B	C
1M Tris pH8.0	3ml	30 mM
1M sodium chloride	20 ml	200 mM
IGEPAL	1 ml	1%
De-ionized water	76 ml

Bead Elution Buffer: 10 ml, store at room temp.

A	B	C
1M Tris pH6.8	1.3 ml	130 mM
Glycerol	1.6 ml	16%
SDS	0.4 g	4%
0.2% Bromophenol Blue	0.5 ml	0.01%
De-ionized water	to 10 ml

Reagents and Equipment

Acetyl-Lysine Affinity beads

Mouse IgG Control beads

beta mercaptoethanol

Microfuge at 4°C

Rotator at 4°C equipped to hold microfuge tubes

Multipurpose mini spin columns (optional)

Each assay requires 50µl of bead suspension. Aliquot the required number of beads into the required number of tubes.

ACETYL-LYSINE IP: IMMUNOPRECIPITATION STEP

Aliquot 50 μl of Acetyl-lysine affinity bead slurry (approx. 50 μg conjugated antibody) for the required number of IP reactions.

Collect beads by centrifugation at 5000 x g for 1 minute and remove supernatant containing acetylated proteins.

Add 2 μl of beta mercaptoethanol to each tube and place at -80°C. These samples are the 24h IP elutions.

Take the supernatants saved in step 2.5 and add a fresh 50 μl aliquot of appropriate beads to each.

Incubate for a further 24h with rotation at 4°C. These will be the 48h IP samples.

24h 0m 0s

Collect the 48h IP beads as above. Lysate supernatants can be discarded.

The 48h IP elutions should be pooled with the corresponding 24h elutions. Samples can now be processed for western blot analysis.

Aliquot 50 μl of mouse IgG control bead slurry (approx. 50 μg of conjugated antibody) for the required number of IP control reactions.

Wash the beads once with 1 ml each of PBS. Collect beads by centrifugation at 2000 x g for 1 minute at 4°C and remove PBS.

Add 1 mg of appropriate lysate to corresponding beads and incubate with rotation (approx. 25-30 rpm) at 4°C for 24h.

24h 0m 0s

Collect beads by centrifugation at 2000 x g, 1 minute, 4°C . Save the supernatants in clearly labeled tubes on ice

Wash the beads by adding 1 ml of Bead Wash Buffer and rotating (25-30 rpm) for 10 minutes at 4°C.

0h 10m 0s

Collect beads as above and repeat the bead wash step 4 more times.

0h 10m 0s

0h 10m 0s

0h 10m 0s

0h 10m 0s

After the final wash, make sure that the wash buffer is completely removed from the beads. A fine bore pipette tip can be used for this step. Failure to remove all wash buffer will reduce effectiveness of the elution buffer and will result in volumes too large for gel analysis.

Elute the acetylated proteins by adding 27 μl of Bead Elution Buffer and incubating at room temp. for exactly 5 minutes. The tubes can be gently flicked to mix the beads and elution buffer. Do not use a pipette for mixing.

Wash beads in Bead Wash Buffer

1. Boil samples for 5 minutes before running on SDS-PAGE.

When the beads are ready, thaw lysates in a room temperature water bath.

WESTERN BLOT: BUFFERS AND EQUIPMENT

Western Transfer Buffer (1L per gel), store at room temp.

A	B	C
Glycine	14.4g	192 mM
1M Tris-HCl pH8.0	25 ml	25 mM
Methanol	150 ml	15%
Deionized water	to 1L final volume

Tris Buffered Saline/Tween (TBST Buffer) (10L). store at room temp.

A	B	C
1M Tris-HCl pH8.0	100 ml	100 mM
5M sodium chloride	300 ml	750 mM
Tween-20	5 ml	0.05%
Deionized water	to 10L final volume

Antibody Block Buffer: 50ml, store at room temp.

A	B	C
TBST pH8.0	50 ml	1X
powdered skimmed milk	2.5 g	5%

Antibody Dilution Buffer: 50ml, store at room temp.

A	B	C
TBST pH8.0	50 ml	1X
powdered skimmed milk	1.5 g	3%

WESTERN BLOT: PROCESS & ANALYSIS

Transfer the proteins to PVDF membrane using the Western Blot Transfer Buffer. Transfer at 20V for 17h.

17h 0m 0s

Block the membrane for 2h in Antibody Block Buffer.

2h 0m 0s

Dilute primary antibody 1:1,500 in Antibody Dilution Buffer and incubate with shaking for 1.5h at room temperature. We use 10 ml of diluted primary antibody for a mini-gel sized blot.

1h 30m 0s

Wash blot 2 x for 5 minutes each in TBST.

0h 5m 0s

0h 5m 0s

Dilute secondary-HRP antibody 1: 25,000 in Antibody Dilution Buffer and incubate with shaking for 40 minutes at room temperature. We use 10 ml of diluted primary antibody for a mini-gel sized blot.

0h 40m 0s

Wash the blot in 50-100 ml of TBST for 10 minutes.

0h 10m 0s

Repeat wash 5 more times.

0h 10m 0s

0h 10m 0s

0h 10m 0s

0h 10m 0s

0h 10m 0s

Develop the blot using Thermo Fisher Super Signal West Dura Chemiluminescent reagent, 10 minute incubation at room temp. in 4ml of reagent per mini gel blot is recommended.

Develop the blot and save the image as a tiff file. Analyze the PD-L1 signal (approximately 50 kD) using ImageJ software.

Centrifuge lysates as soon as they are all thawed. Centrifuge at 4°C in a microfuge at 14,000xg for 5m to ensure any degraded protein is pelleted separately from the sample.

Save a small amount of lysate (~20µl) to run as a western input lysate control. Add 5µl of 5x SDS buffer and save for western analysis at -20°C.

Carefully (so as not to disturb any centrifuged pellet) pipette 1mg of lysate and add to each tube containing beads (both control and acetyl).

Incubate the tubes on a rotating platform at 4°C for 24h.24h 0m 0s

Collect beads by centrifugation at 5000xg for 1m at 4°C.

Aspirate off as much supernatant as possible without disturbing the beads.

Wash beads in 1ml of bead wash buffer (inhibitors are not necessary at this stage) for 10m each on a 4°C rotating platform at mid to high speed.

Complete the wash step four more times (five washes total).

After the final wash, completely remove the buffer supernatant with a pipette. Minimal disruption of the bead pellet is necessary.

Add 28µl of bead elution buffer and resuspend the beads by gently tapping and flicking the side of the tube.

Incubate beads in elution buffer at room temperature for exactly 5m.

Gently pipette each bead suspension to one of the spin columns.

Place the spin column in a fresh collection tube and centrifuge at 10,000xg for 1m at room temperature to collect the IP sample.

Add 2μl of mercaptoethanol to each sample and mix well with a pipette.

Samples can then be stored at -20°C.

Prepare gel box(es) with 1x SDS Gel Running Buffer and 4-20% Tris-glycine Gel(s).

Run gel(s) at 120V for 2h.2h 0m 0s

While the gel is running, make 2L of cold western transfer buffer according to the recipe below.

Place the buffer in the -20°C freezer to cool down.

Get Western blot transfer tank(s) and place them in a cold room to cool down (4°C).

When the gel is completed, soak in ~100mL of cold western transfer buffer for 15m with gentle shaking. This can be done at room temperature.

Cut blot-sized piece(s) of PVDF membrane and soak these for 1m in methanol.

Rinse PVDF membrane(s) briefly in water and transfer them into western transfer buffer until ready to use.

Assemble gels into the western blot apparatus.

Blot gel(s) at 20V for 17 hours (overnight) in a cold room (4°C).

DO NOT DRY BLOT after transfer. Move blot out of apparatus and go directly to block.

Block the blot in 5% milk in TBST. Incubate for 1 hour at room temperature with constant shaking.1h 0m 0s

Rinse briefly in TBST.

Locate the PDL1 antibody and dilute it to a 1:1500 concentration in TBST with 3% milk.

Add 10ml of diluted primary antibody.

Incubate the primary antibody for 1.5 hours at room temperature with constant shaking.

Before removing from the primary antibody, make a 1:25,000 anti-rabbit secondary antibody solution in TBST with 3% milk.

Wash blot(s) 2 times in TBST for 3 minutes

Add 10 ml of diluted secondary antibody.

Incubate the secondary antibody for 40 minutes at room temperature with constant shaking.

Wash the blot 6 times for 10 minutes each in 50mL of TBST.

Develop blots with Super Signal West Dura Ultra HRP reagent. Use 4mL of 1X reagent per blot. Incubate for 5m at room temperature with constant shaking.

Develop western on the following settings for 1m, 5m, and 10m:

• High/Medium Speed/Resolution

• Show Saturation

• No Autoconstrast

Print all pictures and record the date and exposure time on each of them.